This invention relates to the receptors of the NGFI-B family and in particular to compounds that modulate the activity of such family members.
The nuclear receptor (NR) superfamily comprises more than 150 different proteins, most of which are believed to function as ligand activated-transcription factors, exerting widely different biological responses by regulating gene expression (for review, see Di Croce et al, EMBO J1 8:6201-6210 (1999); Mangelsdorf, et al Cell 83:825-839 (1995); Perlmann, et al, Cell 90:391-397 (1997)). Members of this family include receptors for endogenous small, lipophilic molecules, such as steroid hormones, retinoids, vitamin D and thyroid hormone.
In addition, many members of this family lack known ligands and are therefore referred to as “orphan receptors” (for review, see Giguere, et al, Endocrine Rev 20:689-725 (1999); Kastner, et al, Cell 83:859-869 (1995)). During recent years small, lipophilic ligands and activators have been identified for several orphan receptors, leading to new insights into mechanisms of metabolic and regulatory control. These findings have dramatically increased understanding of endocrinology and it's relationship to disease and offer the potential for the development of new classes of drugs able to act on previously uncharacterized signaling pathways, (for review, see Mangelsdorf et al, Cell 83:841-850 (1995); Giguere, et al., supra. Specific examples of orphan nuclear receptors which appear to be specifically regulated by ligands include, without limitation, RXR, FXR, LXR, CAR, and ROR.
In addition a number of orphan nuclear receptors exhibit high constitutive activity, or have poorly defined or lack high affinity ligand binding domains. Examples of orphan nuclear receptors within this class include members of the NGFI-B family, and SHP. Even though such receptors are unlikely to be directly regulated by ligands, interactions with other nuclear receptors, which are ligand responsive, such as, RXR, can confer upon the heterodimer complex ligand responsiveness.
Members of the NGFI-B family and their various alternate names include those listed below.
Receptor name andSubtypeAlternative NamesAccession no.NGFI-B-alpha (Nur77)Nur77, TR3, N10XM_083884NR4A1NAK-1, TIS1NGFI-B-beta (Nurr1)Nurr1, RNR-1NM_006186NR4A2NOT, HZF3, TINUR,TR3βNGFI-B-gammaNOR-1, MINOR, TEC,XM_037370(NOR-1)CHNNR4A3
Nuclear receptors of the NGFI-B family may act on gene expression as monomers, homodimers or heterodimers (with RXR), and each of these entities can bind to different hormone response elements (HREs) and natural promoters. Hormone response elements can contain one or two consensus core half-site sequences. For dimeric HREs, the half-sites can be configured as inverted, (IR) everted (ER) or direct repeats (DR) separated by a variable number of spacer nucleotides.
For monomeric HREs, a single half site is preceded by a 5′-flanking A/T-rich sequence. Half site sequences can deviate quite considerably from the consensus sequences, especially for dimeric HREs in which a single conserved half site is usually sufficient to confer high-affinity binding to the homo- or heterodimer complexes. Naturally HREs rarely contain two perfect consensus half sites. For example NGFI-Bβ, binds to monomeric response elements (NBREs) containing the 5′-extended core motif (AAAGGTCA), and genes shown to be regulated in this manner include tyrosine hydroxylase.
The homodimer binding site consists of two inverted NBREs separated by a 6 base pair spacer and this site confers high responsiveness to gene expression in the presence of NGFI-Bα. Homodimer binding by NGFI-B α, β and γ was also observed on the pro-opiomelanocorticotropin (POMC) gene promoter.
Heterodimers of RXR and NGFI-Bα and β (but not γ) can bind to DR5 response elements and on these elements the heterodimer complex is efficiently activated by RXR ligands. Further it is becoming increasingly apparent that this may be an important physiological mechanism of regulation of NGFI-Bβ mediated gene expression. Additionally, the activity of NGFI-B family members appears to be independently regulated by posttranslational modifications such as phosphorylation that occurs independently of ligand binding to the nuclear receptor or heterodimer complex.
NGFI-Bβ-RXR heterodimers are present in the developing CNS in vivo and naturally occurring RXR ligands accumulate during development and in the postnatal brain consistent with a functional role for NGFI-Bβ-RXR heterodimers in the maintenance of developing and mature neurons.
In mammals three forms of RXR have been identified RXR α (NM—002957), RXR β (XM—042579) and RXR γ (XM—053680). The RXR family is ubiquitously expressed, although individual RXR genes display unique but overlapping patterns of expression during development and in adult tissues.
Known RXR ligands include the vitamin A metabolite 9-cis retinoic acid, and a variety of fatty acid lipid metabolites including the essential fatty acid docosahexaenoic acid (DHA; 22:6n-3). DHA deficiencies lead to neurological abnormalities and diminished learning ability in man (see Gamoh, et al, Neurosci 93:237-241 (1999); Fernstrom, Lipids 34:161-169 (1999); Sheaff Greiner, et al, Lipids Suppl 34:239-243 (1999)). Moreover, dietary DHA may be beneficial in the treatment of atherosclerosis, inflammation and cancer (Horrocks, et al, Pharmacol Res 40:211-225 (1999); Rose, et al, Pharmacol Theraput 83:217-244 (1999)).
Given the ubiquitous expression of RXR, the level of NGFI-Bβ expression may determine the responsiveness of neurons to RXR ligands. NGFI-B family members are highly expressed in the adult nervous system where they are induced as part of the immediate early response to stimuli such as growth factors, membrane depolarization, and seizures. Their-pattern of expression outside the nervous system is broad.
NGFI-Bβ and its highly homologous family members Nur77 and Nor1 can be rapidly induced by various stimuli, including hypoxic/ischemic stress and kainic acid-induced excitotoxicity, (Donaldson et al., Neurosci. Lett (1995) 196(3) 181-4; Schmidt-Kastner et al., Science World J. 2001 1(1 Suppl 3) 95) and recent studies have identified a function for NGFI-Bβ-RXR heterodimers in neuronal survival in response to injury. The use of NGFI-B/RXR specific compounds could thus provide a novel therapeutic avenue for the treatment of stroke and neuronal damage, healing and repair.
The expression of NGFI-B family members has also been associated with the differentiation and survival of dopaminergic neurons generated from embryonic stem cells. (U.S. Pat. Nos. 6,395,546, 6,312,949, and 6,284,534). The use of NGFI-B/RXR specific compounds could thus also provide a novel method of generating dopaminergic neurons for transplantation and for improving the survival of cells once transplanted into the recipient.
Various studies implicate NGFI-Bβ (Nurr1) as a critical regulator of dopamine production in the ventral mid brain, and have demonstrated that NGFI-Bβ mutant mice fail to generate midbrain neurons with a dopaminergic phenotype, (Science (1997) 276 248-250; Proc. Natl. Acad. Sci. USA (1998) 95 4013-4018). Since loss of midbrain dopaminergic neurons is associated with the etiology of Parkinson's disease, the use of NGFI-B/RXR specific compounds could provide a novel therapeutic avenue for the treatment of this disease.
NGFI-Bβ+/− adult mice also show greater susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that elicits Parkinson's disease symptoms, (Le et al., (1999) Exp. Neurol. 159(2), 451-8) suggesting that Nurr1 expression is protective to the development of Parkinson's in adults. Finally NGFI-Bβ polymorphisms are associated with increased risk for Parkinson's disease and diffuse Lewy body disease (Zheng et al., Arch Neurol 2003 60(5) 722-5).
These results suggest that maintaining Nurr1 activity, for example through administering one of the compounds of the present invention may delay or prevent onset of Parkinson's and other neurodegenerative diseases. Accordingly compounds that modulate NGFI-B or NGFI-Bβ/RXR heterodimers would be likely to have utility for use in the treatment, prevention or amoliration of the symptoms of Parkinson's disease.
Mutations in NGFI-Bβ have also been identified in patients with schizophrenia and manic-depressive illness (WO 01/00907), suggesting that compounds that modulate NGFI-B or NGFI-Bβ/RXR heterodimers would be likely to have utility for use in the treatment, prevention or amoliration of the symptoms of schizophrenia and manic depressive illness.
NGFI-Bβ expression in the paraventricular nucleus and adrenal cortex is induced by stress, and ACTH treatment strongly up regulates NGFI-Bα and, expression in the adrenal gland. In addition NGFI-Bα was shown to regulate the steroid 21-hydroxylase (CYP21) and steroid 17-hydroxylase (CYP17) gene promoters (Mol. Cell. Biol. (1993) 13 861-868. The positive action of CRH on the POMC promoter was shown to be modulated via NGFI-Bα and β with the feedback repression of the hypothalamus-pituitary axis (HPA) by glucocorticoids at the level of the pituitary mediated by direct non-productive GR-NGFI-B interactions. Accordingly compounds that modulate NGFI-B or NGFI-B/RXR heterodimers would be likely to have utility for use in the treatment, prevention or amoliration of the symptoms of HPA dysfunction.
The induction of the genes encoding the NGFI-B family members by pro-inflammatory cytokines suggested a role for this family in mediating inflammatory responses. Indeed recent studies have suggested that these receptors are expressed at sites of chronic inflammation such as the synovium of patients with arthritis and in human atherosclerotic lesions (WO 01/87923). Accordingly compounds that modulate NGFI-B/RXR heterodimers would be likely to have utility for use in the treatment, prevention or amoliration of the symptoms of inflammatory responses including arthritis and in human atherosclerotic lesions.
Over expression of NGFI-Bα in smooth muscle cells inhibits cell proliferation and is protective in a transgenic mouse model of atherosclerosis while a dominant negative receptor has the opposite effects (EMBO. J. (1997) 16 (8) 1865-75). Thus members of the NGFI-B gene family are linked to the control of cell proliferation and may function as natural brake to dampen acute inflammatory reactions.
Retinoic acids and other retinoids exert anticancer effects through the retinoid receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Recently it has been demonstrated that the anticancer effects of retinoids on MDA-MB-231 breast cancer cells were mediated by RXR-NGFI-B family heterodimers, (Mol. Cell. Biol. (1997) 17(11) 6598-608). Accordingly compounds that modulate NGFI-B or NGFI-B/RXR heterodimers would be likely to have utility for use in the treatment, prevention or amoliration of cancer.
Although single genetic knockouts of NGFI-B α and γ appear normal, double knockouts of compound heterozygotes (NGFI-B α−/− NGFI-B γ and NGFI-B α+/−NGFI-B γ−/−) develop myeloid leukemia once again coupling the NGFI-B to cell proliferation and immune cell function. Accordingly compounds that modulate NGFI-B or NGFI-B/RXR heterodimers would be likely to have utility for use in the treatment, prevention or amoliration of the symptoms of acute inflammatory reactions.